The Trp53 delta proline (Trp53ΔP) mouse exhibits increased genome instability and susceptibility to radiation-induced, but not spontaneous, tumor development

Abstract

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.

Keywords: Trp53; cancer radiation; genomic instability.

Figure 1. Tumor-free survival and spectrum

a) Kaplan-Meier curve shows overall survival in days of Trp53ΔP homozygous mice on a mixed 129/SvJ and C57BL/6J background exposed to 4Gy γ radiation (n=36, red line), versus sham-irradiated control mice, (n=20, blue line) (P<0.0001, log-rank test). b) Pie chart shows types of tumors developed post-irradiation based on differentiation; lymphoid (12 of 36), mesenchymal (16 of 36) and epithelial (8 of 36). c) Further details of tumor category, type and involved organ with the frequency of occurrence indicated. BA = bronchio-alveolar.

Figure 2. Radiation induces a broad tumor spectrum in a wide range of tissues

a) Hematoxylin and Eosin (H&E) of a range of tumor types developed in irradiated Trp53ΔP mice, magnification is 20×, scale bar represents 200µm. i) ID:124- lymphoma infiltrating liver ii) ID:127- lymphoma infiltrating lung. iii) ID:151- hemangiosarcoma of the intestinal stroma. iv) ID:169- hemangiosarcoma invading the spinal cord, surrounding bone and of soft tissues. v) ID:166- papillary bronchio-alveolar adenoma of the lung. vi) ID:162- adenocarcinoma of the mammary gland. b) Representative H&E of two angiosarcomas showing vascular clefts lined by neoplastic endothelial cells, and, IHC for CD31 of both angiosarcomas showing strongly positive membrane staining for CD31. ID numbers corresponding to Table 1 are indicated, magnification is 20×. Scale bar represents 100 µm. c) Scatter graph depicting days survival post-irradiation of various tumor types; bars indicate p values as determined by unpaired Student’s t-tests, *=<0.05 **=<0.008 ***=<0.0001, error bars represent 95% confidence interval. d) Representative CD3 and B220 IHC staining of two lymphomas. ID:128 is a thymic lymphoma, which stains positive for CD3 and is interpreted as a T-cell lymphoma. ID:124 is a lymphoma in the liver and has a mixed cell infiltrate. The majority of cells with large dysplastic nuclei are positive for B220 staining indicating origin as a B-cell lymphoma. ID numbers correspond to those in Table 1, magnification is 20×. Scale bar represents 200µm.

Figure 3. Abnormal centrosomes can participate in formation of multi-polar spindles in Trp53ΔP MEFs and result in increased aneuploidy population

a) Flow cytometric analysis of cell cycle distribution by DNA content of MEFs of genotypes indicated. The average % of cells >4n (aneuploidy population) is 21.71 (WT), 34.43 (Trp53ΔP) and 40.20 (Trp53−/−). b) Representative image of immunostaining performed in MEFS of genotypes WT, Trp53ΔP, and Trp53−/− 24 hours 4Gy γ irradiation. Centrosomes are stained with pericentrin (green), mitotic spindles stained with alpha-tubulin (red). Nuclei were visualized with DAPI staining. All images are merged in the bottom panel. c) Quantification of the fraction of cells with >2 centrosomes per cell as determined by gamma tubulin and pericentrin staining. MEFs of genotypes WT, Trp53ΔP, and Trp53−/− were exposed to 4 Gy irradiation or sham irradiated and harvested at time points indicated. Studies were performed at minimum in triplicate. The mean number of cells counted per genotype/timepoint was 293.

Figure 4. Centrosomal abnormalities in thymus

a) Representative confocal images of centrosome staining in thymus from mice 24 hrs after irradiation with 4 Gy γ radiation. Centrosomes were co-stained with pericentrin (green) and gamma-tubulin (red), DAPI was used to visualize nuclei; scale bar represents 10µm. b) Quantification of the fraction of cells with >2 centrosomes in thymus tissue. For each genotype and timepoint 2–3 mice were used and the mean number of cells counted was 609.